The pathogenic bacteria classified as Streptococcus pneumoniae (pneumococci, Pn) have been subdivided into 84 antigenic serotypes, based on the capsular polysaccharide (Pn-Ps) of the organism. Disease states attributable to these organisms include pneumonia, meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis and conjuctivitis. The preponderance of these diseases, however, are caused by a limited subset of the 84 known isolates. Thus a polyvalent vaccine containing the Pn-Ps from the most prevalent and pathogenic isolates of the organism can provide protection against a very high percentage of the most frequently reported pathogens of this class.
Polyvalent vaccines have been produced that are efficacious in raising protective immune responses against the pneumococci in adults. "PNEUMOVAX.RTM. 23" (Pneumococcal Vaccine Polyvalent, MSD; see PDR, 1990 edition, p. 1431), for example, is a liquid composition containing 50 .mu.g/ml of each of the 23 different, unconjugated pneumococcal polysaccharides, all of which are on deposit with the ATCC and provid one possible source of starting material for this invention. "PNEUMOVAX.RTM. 23" comprises each of the following free, that is unconjugated, polysaccharides: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F, accounting for about 90% of pneumococcal blood isolates. However, such vaccines are least effective in the segment of the population most at risk for pneumococcal infections: B-Cell immunocompromised individuals, the elderly and infants younger than two years old who depend on T-cell responses for immune protection. Since unconjugated polysaccharides are poor inducers of T-cell immune responses, conversion of the Pn-Ps into immunogens capable of inducing T-cell responses is the key to producing adequate protection in this target population. Use, however, is not restricted to this group of individuals. For example, administration of a vaccine, comprising one or more of the novel conjugates, to a female mammal prior to or during pregnancy raises antibodies in the mother which can passively protect a developing fetus and suckling infant even though the vaccine is not administered directly to the fetus or infant. Such conjugate vaccines should also prove useful for eliciting antibodies for ultimate passive protection of at risk populations, such as newborns or siblings of infected individuals.
Polysaccharides found to be poorly immunogenic by themselves have been shown to be quite good immunogens once they are conjugated to an immunogenic protein, PRO, [Marburg et al., U.S. Pat. No. 4,695,624; Schneerson et al., New Dev. with Hum. & Vet. Vaccines, 77-94 (1980); Schneerson, et al., J. Exptl. Med., 152, 361 (1980); Anderson, Infection and Immunity, 39, 233 (1983)]. However, a major problem in the production of such conjugates is the viscous and non-homogenous nature of the polysaccharide starting material and hence the difficulty in defining the conjugate product chemically. Thus, a process is required wherein the starting materials are as well defined as possible and each step in the synthetic route is assayable as to intermediate formed. The process herein disclosed satisfies this requirement by providing highly immunogenic conjugate immunogens against the cognate pathogens from which the Pn-Ps is derived. The conjugates are useful in infants younger than two years old.
Marburg et al., [J. Am. Chem. Soc., 108, 5282 (1986), and U.S. Pat. Nos. 4,695,624; 4,830,852; 4,882,317] disclosed a means of conjugating polysaccharides and immunogenic proteins through bigeneric spacers. The PRO was derivatized to exhibit pendant nucleophilic or electrophilic groups (PRO*), while a partner Ps was functionalized so as to exhibit pendant groups of opposite reactivity (Ps*). Upon combining Ps* with PRO*, bigeneric spacers were formed, covalently joining Ps to PRO (Ps-PRO). Upon acid hydrolysis, the bigeneric spacer is released as an unusual amino acid, quantitated by amino acid analysis, and thereby providing a means of proving covalency.
This invention discloses a process improved over that which is disclosed in U.S. Pat. Nos. 4,695,624; 4,830,852; 4,882,317. The improvements include preparation of Pn-Ps starting material having more specific, reproducible and manageable physical properties than provided by crude Pn-Ps preparations, including: increased solubility, increased filterability, increased purity (reduction in contamination with group specific C-polysaccharide (C-Ps), and reduced molecular weight, polydispersity and viscosity. The resultant conjugates disclosed herein are improved over those provided by the U.S. Pat. No. 4,695,624 process with respect to increases in the consistency and ease of preparation, improved antigenicity and improved purity of the final product. Especially significant is the 3-20 fold reduction of group specific C-polysaccharide and peptidoglycan in the pre-conjugation Pn-Ps relative to the pre-conjugation Ps preparations of the U.S. Pat. No. 4,695,624 patent. Although the presence of the C-polysaccharide contaminant does not interfere with the immune responses against the type specific antigens, C-Ps could participate in the conjugation reaction rendering it less specific and controlled for the type-specific Ps. Furthermore, production of anti-C-polysaccharide antibodies may correlate with the tissue destruction observed in some unresolved pneumococcal infections.
In addition to the novel conjugate product, this invention discloses a new process for preparing partially hydrolyzed, highly purified pneumococcal polysaccharide intermediates, novel compositions comprising from one to ten different conjugates and methods of using the invention. Of particular interest are the capsular polysaccharides included in "PNEUMOVAX.RTM. 23" (Pneumococcal Vaccine Polyvalent, MSD; see PDR, 1990 edition, p. 1431). A most preferred subset are the capsular polysaccharides of Streptococcus pneumoniae subtypes 6B, 23F, 19F, 14, 18C, 4 and 9V, as this small group of pneumococcal subtypes are estimated to be responsible for between 75-85% of pneumococcal infections in infants and children. The methods provided herein are applicable to a broad collection of pneumococcal and other bacterial polysaccharides.